1. Field of the Invention
The present invention relates to method and apparatus for separating cellular constituents of a whole blood and, more particularly, for separating by centrifugation peripheral blood lymphocytes to achieve high purity and yield.
2. Description of the Prior Art
The clinical evaluation of biological fluids, such as blood, is a vital part of medical practice. Such evaluation is made in respect of particular constituents of the biological fluid and provides an indication of the medical state of the donor.
Once the blood sample is collected, it is often necessary that the particular constituent of the blood sample to be analyzed is available to the technician in as a pure form as possible. Often, centrifugation is used to separate such constituent. During centrifugation, the blood is "spun-down" in a container for a predetermined time, until all suspended erythrocytes, leukocytes and platelets are separated from the plasma or serum. The serum can be evaluated in respect of clinically significant soluble constituents present therein. Following centriguation, the separated serum and cells (and platelets) are separated, either by decanting, siphoning, or pipetting. The serum can be analyzed, e.g., by automated analytical systems as described in U.S. Pat. No. 4,321,432 which is assigned to the instant assignee, to determine the concentration of selected constituents therein, e.g., glucose, cholesterol, etc. Alternatively, the separated cells and platelets can be evaluated, providing clinically significant information. For example, the absolute and relative number of different kinds of white blood cells (leukocytes) and, also, the platelets are a source of vital information concerning the medical state of the donor. Commonly, separation of leukocytes is achieved by centrifugation, wherein a physiologically inert barrier having a specific gravity between the respective specific gravities of the leukocytes and erythrocytes is initially introduced into a tube containing the blood sample. Accordingly, during centrifugation, the leukocytes, along with the supernatant, and erythrocytes are separated by the barrier. As the respective specific gravities of the different subsets of leukocyctes are similar, the separation of pure lymphocytes, a subset of the leukocytes, is difficult to obtain by this technique. Such technique is described, for example, in U.S. Pat. No. 4,190,535.
As hereinafter employed, purity may be understood to be equal to the number of lymphocytes present in the supernatant after separation divided by the total number of all kinds of leukocytes present in that fraction of blood sample. Yield may be understood to be equal to the total number of lymphocytes of the separated sample divided by the number of lymphocytes in the original whole blood sample. Viability may be understood to be equal to the number of Trypan blue excluding lymphocytes in the separated sample divided by the total number of lymphocytes in such separated sample.
There is a growing need, both in the research and clinical laboratory, for obtaining pure peripheral blood lymphocytes. An evaluation of the lymphocyte population in a blood sample provides information of great clinical significance. For example, in histocompatibility typing, the reaction of lymphocytes to certain reagents (antigens) is useful in determining whether a human organ will be accepted or rejected when transplanted. Also, in a immuno-competence testing, lymphocytes are mixed with particular substances (antigens) and the reaction therebetween noted. The degree of reaction provides an indication of the presence of an immuno-deficiency disease, of an auto-immune disease, etc., in a patient. However, to obtain a meaningful evaluation, it is essential that the lymphocytes obtained from a blood sample be highly pure, i.e., void of the presence of other subsets of leukocytes, and accurately reflect the total population of lymphocytes in the original blood sample. A high degree of yield and purity is essential for the evaluation to be truly representative of the medical condition of the patient.
Numerous techniques are known in the prior art for separating lymphocyctes. For example, in U.S. Pat. No. 3,709,791, assigned to the instant assignee, method and apparatus are described for separating lymphocytes from other cellular constituents in a whole blood sample. As described, the blood sample is mixed with magnetic particles in a disposable syringe and incubated, whereby the particles are phagocytized by monocytes and neutrophils but not by lymphocytes. In effect, the phagocytic leukocytes are "magnetically tagged"; also, a sedimentation agent is introduced into the syringe to promote sedimentation of the erythrocytes. Subsequently, the blood sample, less the sedimented erythrocytes, is forced from the syringe along the conduit which extends through a high-strength, non-uniform magnetic field. Accordingly, the tagged leukocytes and free magnetic particles are retained in the conduit by such magnetic field, allowing collection of the purified lymphocytes.
Also, centrifugation techniques have been used to separate leukocytes. One widely used technique is described, for example, in Transplantation, Vol. 22, No. 21, p. 101 (1976). In practicing such technique, a diluted blood sample is layered onto a Ficoll-Hypaque liquid, which is used as a density barrier and has a controlled specific gravity intermediate the respective specific gravities of the mononuclear leukocytes (and also plasma and platelets) and the erythrocytes and many segmented leukocytes. As the Ficoll-Hypaque is an aqueous liquid, the diluted blood sample must be carefully layered onto the surface thereof, so as to prevent mixing therebetween. Again, because the supernatant and cellular constituents are miscible with the Ficoll-Hypaque, separation of the phases following centrifugation must be again carefully effected. Generally, the sample is diluted (1:1 to 1:4), and the yield obtained is relatively low, at least in part, due to subtle effects of dilution. Also, as the respective specific gravities of the subsets of leukocytes are similar, the purity of separated lymphocytes is low.
Attempts have been made to improve separation of lymphocytes by diluting the sample prior to centrifugation, so as to obtain a somewhat sharper banding of cells at the density barrier/diluted plasma interface. This sharper banding facilitates the harvesting and somewhat increases the lymphocyte yield. However, the yield and purity of the harvested lymphocytes remain at a level which is less than ideal.
Also, advances have been made in respect of density barrier materials useful in centrifugal separation techniques. For example, U.S. Pat. No. 4,021,340 and U.S. Pat. No. 4,333,564 describe the use of hydrophobic gel-like, inert compositions having thixotropic properties. Such materials have certain advantages over Ficoll-Hypaque, in that the introduction of sample into the separating container is facilitated. Because such materials are hydrophobic, there is almost no tendency for the sample, when introduced, to be intermixed with such material. In addition such materials are relatively easily deformed and displaced by centrifugal forces but provide following separation, a relatively undeformable barrier which greatly facilitates separation of the supernatant.